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Study Of The Inhibition Capacity Of Maillard Reaction Products Derived From Cysteine On Tyrosinase-catalyzed Enzymatic Browning

Posted on:2018-12-29Degree:DoctorType:Dissertation
Country:ChinaCandidate:H N XuFull Text:PDF
GTID:1311330518486524Subject:Food Science
Abstract/Summary:PDF Full Text Request
Enzymatic browning exists widely in nature.Most enzymatic browning occurring in food industry is detrimental to food quality,including reducing nutritional value,influencing organoleptic properties of food,etc.Enzymatic browning has a remarkable effect on fresh cut fruits and vegetables,affecting negatively the shelf life of fresh cut produces.For anti-browning agents now in wide use,some of them are limited in application range due to the safety problem,or some of them show low inhibitory efficiencies,or some of them would be consumed during the inhibition process.So,looking for effective anti-browning agents has always been a hotspot.Until now,there are only limited studies on the enzymatic browning inhibition capacity(BIC)of Maillard reaction products(MRPs)and its inhibition mechanism remains unclear.Reactant types as well as processing parameters of Maillard reaction all have significant effects on the constituent and property of MRPs.In this study,the BIC,tyrosinase inhibition capacity(TIC)and antioxidant capacity of MRPs prepared at various conditions was systematically investigated in order to elucidate the enzymatic browning inhibition mechanism of MRPs,find the effective species among various MRPs as well as the main active components in MRPs responsible for inhibiting tyrosinase activity,reveal the relationship between the BIC and Maillard reaction processing parameters of MRPs.Furthermore,the BIC of MRPs in real food system was investigated preliminarily in order to provide theoretical basis and technical guidance for its regulation study as well as its practice use in food process and storage.The main contents are as follows:The BIC of MRPs derived from twelve representative amino acids and three sugars(xylose,fructose,glucose)were investigated and its relationship with antioxidant capacity was also studied.The MRPs of Cys,cystine,Arg and His showed higher BIC compared with other amino acids.MRPs from cystine also showed higher BIC,which may be due to the formation of free Cys during Maillard reaction.Lys-MRPs showed the highest absorbance value at 420 nm but with very limited BIC,while Cys-MRPs showed the highest BIC and the lowest absorbance value at 420 nm.For MRPs from the twelve amino acids with glucose or fructose except Lys and Tyr,A420 can roughly reflect the level of BIC of MRPs.MRPs from Tyr showed the most potent antioxidant capacity but very limited BIC,while Cys-MRPs showed both higher antioxidant capacity and BIC compared with other amino acids.For MRPs from twelve amino acids with glucose or fructose except Lys,Cys and Tyr,correlation analysis showed that there was a moderate and positive correlation between BIC and Fe2+ reducing power.The type of amino acids,initial pH,temperature and time of the Maillard reaction were found to greatly influence the BIC and antioxidant capacity of the resulting MRPs.The suitable pH for generating efficient browning inhibition compounds varies depending on different amino acids: acidic pH was favorable for Cys,while for His and Arg,neutral and alkaline pH were suitable,respectively.Increasing heating temperature and time in a certain range could improve the BIC of MRPs of Cys,His and Arg,while further increasing will deteriorate their browning inhibition efficiencies.There is no clear relationship between BIC and antioxidant capacity of MRPs when reactant type and processing parameters of Maillard reaction are variables.Afterwards we investigated the impact of chemical polarity and molecular weight of Cys-MRPs on its BIC,TIC and antioxidant ability(including DPPH radical scavenging ability and copper chelating ability).The results showed that the active compounds with TIC in Cys-MRPs were non-polar biased while multiple components with various polarities contributed to the BIC of MRPs.Results showed a significant difference between TIC and BIC of Cys-MRPs with different polarity or molecular weight.In addition,some components in Cys-MRPs possessing high DPPH radical scavenging ability might contribute to the overall BIC,but there was no correlation between the DPPH radical scavenging ability and TIC.The copper-chelating ability of different polarity fractions was positively correlated with their corresponding TIC,implying that copper-chelating ability could be used as an indicator of the anti-browning ability of various MRPs.A highly active fraction M5 with TIC was obtained,which showed noncompetitive inhibition on tyrosinase.The inhibition constant of M5 was 3.10 ?g/mL,almost 80-fold of original Cys-MRPs.GC–MS analysis revealed that 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one(DDMP)and 2,5-dimethyl-4-hydroxy-3(2H)-furanone(DMHF)were two major compounds in fraction M5,among which,DMHF showed no inhibitory activity,while DDMP was a noncompetitive inhibitor on tyrosinase and its inhibition constant was 4.99 ?g/mL.Cys-MRPs,which possessed the strongest TIC,was chosen for further study into the inhibition mechanism.Cys had no inhibitory effect on tyrosinase activity,but it can react with the initial product of enzymatic browning process o-quinone preventing the formation brown pigments.Polargraphic and spectrophotometric method were used together in order to get more information about inhibition pattern of Cys-MRPs on mushroom tyrosinase.The results showed that Cys-MRPs can both have the ability to inhibit enzyme activity and react with o-quinone to prevent the occurrence of browning in both a direct and an indirect way.The inhibition pattern and capacity of Cys-MRPs are greatly influenced by Maillard reaction processing parameters.When inhibitor concentration was 0.052 mM,Cys-MRPs derived from optimum conditions could completely inhibit tyrosinase activity.Diafiltration experiments showed that Cys-MRPs have a partially irreversible inhibitory activity on tyrosinase.Supplement of CuCl2 after preincubation treatment with Cys-MRPs can restore its part activity,revealing that the TIC of Cys-MRPs could partially contribute to its copper ion chelating activity.Competitive inhibitors dithiothreitol and kojic acid can protect the enzyme from Cys-MRPs inactivation,suggesting that the acting site of Cys-MRPs on tyrosinase locates at its active site.The correlation between tyrosinase inhibition ability,volatile compounds,non-volatile compounds(HMF,DDMP and maltol)and Maillard reaction conditions of Cys-MRPs was analyzed by partial least square regression(PLSR).3-ethyl-2-formylthiophene,?-dimethylformylthiophene,2,6-dimethylpyrazine,ethylpyrazine,2-ethyl-6-methylpyrazine,2-methyl-3-(2-thienyldithio)thiophene and furfural showed significant and positive contribution to inhibition ability,while 2-propionylfuran and ?-dimethyl-2-formylfuran showed significant but negative correlation with inhibition ability.Among the three non-volatile compounds analyzed only 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one(DDMP)showed significant and positive correlation with the inhibition ability while HMF and maltol showed weak negative correlation.So,the content of non-volatile DDMP in the Maillard reaction system can be used as an indicator of TIC of Cys-MRPs.The reaction temperature and time showed significant and positive correlation with inhibition rate while ratio of sugar to amino acid showed negative effect within the experimental range.Enzymatic browning inhibition capacities of inhibitors are greatly influenced by food matrix and type.Cys and Cys-MRPs had an opposite effect on inhibiting browning of potato juice compared with potato slices,among which potato juice was closer to model system when Cys-MRPs showed stronger browning inhibition capacity than Cys.Cys was the most effective in reducing total color difference change of potato slices,followed by Cys-MRPs and citric acid,and there existed an obvious synergistic effect between Cys and Cys-MRPs;Cys and Cys-MRPs showed the same inhibitory effect on maintaining the colorimetric value of potato slices,both of which were more efficient than citric acid.Cys showed no inhibitory activity on tyrosinase,and its content decreased during the inhibition process,so its inhibitory activity on enzymatic browning is temporary.However,Cys-MRPs can directly inhibit PPO activity.So,it can permanently inhibit the occurrence of enzymatic browning.All in all,Cys-MRPs is a potential anti-browning agent.
Keywords/Search Tags:Maillard reaction products, enzymatic browning inhibition, polyphenoloxidase, tyrosinase, antioxidant
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